根据CD4立体构型设计的新型免疫抑制物J2抑制小鼠胰岛移植急性排斥反应的初步观察

目的评价基于CD4立体构型设计的新型免疫抑制物J2对小鼠胰岛移植后急性排斥反应的影响。方法将900～1000个DBA/2（H-2^d）小鼠的胰岛移植在糖尿病模型C57BL/6（H-2^b）小鼠肾被膜下。移植前用台盼蓝和双硫腙染色检测胰岛细胞活性和纯度。将C57BL/6小鼠随机分为5组,每组10只,在胰岛移植后第1～10天腹腔内注射J2,每天一次。对照组注射生理盐水、CsA组注射环孢素A（10 mg/kg）;实验组J2A组（1mg/kg）、J2B组（4 mg/kg）、J2C组（8 mg/kg）。术后监测不同时间各组小鼠的血糖变化及移植胰岛的存活时间,并观察移植胰岛在术后第三天及发生急性排斥反应时的组织病理学及免疫组化表现。结果获得的胰岛细胞的活性＞95%,纯度＞85%。胰岛移植术前各组糖尿病小鼠的血糖平均高于20.0mmol/L,术后前四天各组小鼠的血糖下降明显,均低于11.1 mmol/L,且各组之间比较差异无统计学意义（P＞0.05）。移植第7天以后,对照组、J2A组小鼠的血糖较术后前四天明显升高（P＜0.05）。术后第八天开始,对照组、J2A组小鼠血糖升高平均高于11.1 mmol/L;CsA组、J2B组、J2C组的血糖仍低于11.1 mmol/L,三组之间无明显差异,但明显低于对照组和J2A组（P＜0.05）。病理学检查示移植后第三天各组移植的胰岛细胞轻度水肿,形态大致正常;移植后第八天对照组、J2A组的移植胰岛出现急性排斥反应病理改变,其他组病理未见排斥表现,小鼠胰岛素抗体免疫组化结果显示胰岛素阳性。CsA组（21.6±2.1 d）、J2B组（19.0±2.7d）、J2C组（18.7±2.3d）的移植胰岛存活时间比较差异无统计学意义,但分别较对照组（8.1±0.56d）、J2A组（8.3±0.48d）显著延长（P＜0.01）。结论一定剂量的J2具有和CsA相似的免疫抑制作用,可明显抑制小鼠胰岛移植后排斥反应的发生,显著延长移植物的存活时间。     

羟基荧光素二醋酸盐琥珀酰亚胺脂标记大鼠肝脏NK细胞体内转输示踪

目的 利用羟基荧光素二醋酸盐琥珀酰亚胺脂(CFSE)标记大鼠肝脏NK细胞进行体内示踪,观察其在受鼠体内的存活.方法 经SD大鼠门静脉转输1×106个/ml CFSE体外标记新鲜分离的SD供鼠肝脏NK细胞,于输人后1、3、7、15 d分别切取肝脏和脾脏行冰冻切片免疫荧光观察,并收集门静脉血监测淋巴细胞和中性粒细胞的比例变化.结果 CFSE对大鼠肝脏NK细胞的标记率为98.63%;转输NK细胞后第1天受鼠肝脏内CFSE荧光最强,7 d时荧光变微弱,15 d时荧光基本消失;脾脏在第1、3天时可见较弱荧光,7 d时荧光消失.转输NK细胞后相同时间点肝脏内CFSE荧光均明显强于脾脏.转输肝脏NK细胞的受鼠门静脉血中性粒细胞比例第1天时最高,随时间延长逐渐下降,第15天时基本回复正常水平,淋巴细胞比例仅一过性降低.结论 转输的肝脏NK细胞在受体内的数量随时间延长而减少,在受体内的存活时间约为15 d.     

免疫调节剂在外科感染及大手术中的作用机制探讨

目的 了解免疫调节剂胸腺肽α-1在外科感染及大手术中的作用机制。方法 分别选择60例外科感染病人和60例胰腺癌根治手术病人，分用药组和对照组。用药组病人在手术后使用胸腺肽α-1，对照组不使用免疫制剂。观察临床疗效、内毒素和细胞因子(IL-2、IL-6、IL-10和TNF-α)、T淋巴细胞亚群CD3^ 、CD4^ 、CD8^ 和NK细胞百分率的变化。结果 外科感染及胰腺癌病人无论用药组和对照组术后内毒素、IL-6和TNF-α均呈下降趋势，IL-2和IL-10呈上升趋势，但用药组大部指标在术后4d即达显著差异，术后7d变化趋势更为明显。外科感染及胰腺癌病人用药组术后CD4^ ／CD8^ 比值和CD4^ 百分率上升；同类病人术后用药组和对照组比较CD3^ 、CD4^ 百分率和CD4^ ／CD8^ 比值较高。用药组临床疗效略高于对照组。结论 胸腺肽α-1在外科感染及重大手术中可提高免疫功能，有利于病人恢复。     

Subclinical impairment of distal renal acidification induced by low-dose cyclosporin A therapy

Twenty-nine psoriasis patients on 5 mg/kg cyclosporin A (CyA) therapy were studied for 3 months using the furosemide test. Five of them (17 %) showed an abnormal renal acidification capacity after furosemide administration: The urinary pH did not sink under 5.3 after furosemide, while the ammonium and titrable acid levels were significantly low. There were no significant differences from controls regarding the serum potassium or fractional potassium excretion. Nevertheless, the transtubular potassium gradient was lower in patients with an abnormal furosemide test result. We conclude that some patients treated with a low dose CyA therapy developed an abnormality in the distal tubular acidification.     

Prolongation of heart allograft survival in rats by interferon-specific antibodies and low dose cyclosporin A

Interferons (IFNs) are important cytokines which exhibit antiviral, antitumor, anticellular, as well as immunoregulatory activities [1]. Among these multiple activities, IFNs are potent inducers of MHC antigen expression of a great variety of cells [2–4], helper and maturation factors in B-cell antibody production [5], and macrophage function [6]. IFNs may therefore play a critical role in triggering antigen recognition and allograft rejection. Cyclosporin A (CyA) is a potent immunosuppressor which selectively inhibits helper T-lymphocyte proliferation in response to alloantigen presentation [7, 8]. CyA has been reported to inhibit interleukin 2 and IFNγ production by helper T lymphocytes [9–11]. In addition, CyA may induce monocyte production of prostaglandin E2 [12], which then reduces MHC class II expression on endothelial cells, monocytes, and macrophages [13]. However, the clinical use of CyA is plagued by its toxic (in particular nephrotoxic) side-effects. These toxic effects are clearly dose-related. It may be very important to develop new products which can act synergistically with CyA to inhibit lymphokine production. The aim of this study was to investigate the effects of combined IFN-specific antibodies and low dose CyA on cardiac allografts in inbred strains of rats.     

外源性IL-2对小鼠CD4^＋CD25^＋调节性T细胞影响的实验研究

目的：探讨IL-2对CD4＋CD25＋调节性T细胞（Tregs）的增殖及功能的影响。方法：提取B6小鼠脾脏细胞,流式细胞仪分离CD4＋CD25＋Tregs,将新鲜分离的CD4＋CD25＋Tregs与抗CD3单克隆抗体、同种同系抗原递呈细胞（APCs）及外源性IL-2共同培养,测定其增殖活性;并检测体外扩增后的CD4＋CD25＋Tregs的免疫抑制活性及其Foxp3的表达。结果：与外源性IL-2共同培养的CD4＋CD25＋Tregs增殖程度强烈,与对照组比较,差异有统计学意义（P〈0.05）;体外扩增的CD4＋CD25＋Tregs抑制CD4＋CD25-T细胞增殖活性的能力与新鲜分离的CD4＋CD25＋Tregs相似（P〉0.05）。体外扩增的CD4＋CD25＋Tregs的Foxp3表达与新鲜分离的CD4＋CD25＋Tregs亦相似（P〉0.05）。结论：外源性IL-2能够消除CD4＋CD25＋Tregs的无反应状态,且体外扩增的CD4＋CD25＋Tregs保持了其抑制活性。     

New strategies in immunosuppression

This article reviews recent papers relating to immunosuppressives and attempts to categorise the bewildering number of new reagents according to their effects on the immune response. It is apparent that the majority of groups are concentrating on reagents which, like cyclosporin A, are predominantly directed at T cells (42% of the last 200 papers inTransplantation, Transplant International andTransplantation Proceedings). The major change in strategy which is occurring relates to the rapidly increasing use of reagents directed against T cell subsets, especially those directed against the interleukin-2 receptor and CD4-positive T cells. This groups's share of the market has risen from 2% to over 12% within 5 years. New successful monoclonal antibodies include reagents directed against antigen-presenting cells and against molecules directly involved in cell adherence. The use of donor bone marrow or subsets of cells from donor bone marrow as inducers of non-reactivity, especially to solid organ grafts, is certainly one of the most exciting of the non-antibody protocols. It is encouraging that relatively specific immunosuppression can be induced in animals by combinations of specific and non-specific reagents as well as by specific reagents alone. This will facilitate the introduction of specific protocols into the human situation, and this strategy holds out great hope for the future. Unfortunately, one of the most effective ingredients of such combination therapies in animal models (anti-CD4) appears to have its tolerogenic potential abrogated by cyclosporin A and FK-506. One area of immunosuppression which is receiving scant interest relates to antibody (especially non human leucocyte antigen antibody) production, but as the interest in xenotransplantation increases it is anticipated that major new interest will emerge in this area.     

Effect of LS-2616 on the graft protection achieved by cyclosporin A,prednisolone, and 15-deoxyspergualin in heart-transplanted rats

The immunostimulator LS-2616 abolishes the effect of cyclosporin A in a rat cardiac transplantation model. The present paper compares the characteristics of rejection obtained under different immunosuppressive regimens with and without additional LS-2616 application in the same model. Cyclosporin A (CyA, 10 mg/kg daily), prednisolone (15 mg/kg daily), or 15-deoxyspergualin (2, 5, or 10 mg/kg daily), all given from the day of transplantation until day 9, protected the grafts during the treatment period. The addition of LS-2616 (160 mg/kg, day —1 until stop) resulted in a total abrogation of the immunosuppressive effect of CyA and prednisolone. However, LS-2616 could only partially or not at all reverse the effect of 15-deoxyspergualine. These results show a certain drug selectivity of LS-2616 in promoting rejection of immunosuppressed allografts. Further studies with LS-2616 may be of benefit in evaluating the mode of action of different immunosuppressive compounds and, thus, contribute to finding more effective antirejection therapies.     

The immunomodulatory effects of a novel agent, leflunomide, in rat cardiac allotransplantation

Abstract We assessed the immunomodulatory effect of leflunomide (LEF) in a heterotopic abdominal model of rat heart transplantation using a major histocom-patibility mismatch (DA X LEW). The endpoint of this study was cardiac rejection assessed by abdominal palpation of the ventricular impulse and confirmed by laparotomy and histology. In this study, LF was investigated using four dosages (5, 10, 20 and 30 mg/kg per day orally) against cyclosporine (CsA) (15 mg/kg per day orally) and FK506 (1 mg/kg per day orally). The ability of LEF to prevent rejection and reverse ongoing acute rejection was assessed. The results showed that untreated hearts were fully rejected by day 5 and that LEF at 5 mg/kg was significantly better than any other dose tested, was superior to FK 1 mg/kg, and was as effective as CsA 15 mg/kg in preventing rejection after a short course of treatment. After a longer course, 10 and 20 mg/kg LEF proved more effective than 5 mg/kg in controlling rejection and as efficacious as 1 mg/kg FK and 15 mg/kg CsA. In the control of ongoing established early rejection. LEF proved to be equally effective, even at lower doses (5 mg/kg), to CsA 15 mg/kg and FK 1 mg/kg. In the control of ongoing established late rejection, 20 mg/kg LEF proved to be superior to 10 mg/kg LEF and 15 mg/kg CsA, and was as effective as FK 1 mg/kg. However, when higher doses of CsA (25 mg/kg) and FK (2 mg/kg) were tested against 20 mg/kg LEF in this mode of rescue, LEF proved as effective as CsA and superior to FK. In the assessment of drug toxicity using body weight as a parameter, 20 mg/kg LEF proved safer than any other LEF dose tested, and safer than 15 mg/kg CsA and 1 mg/kg FK in both short- and long term administration. We conclude that LEF is a relatively safe and potent immunosuppressant with promising clinical potential.     

RNA干扰阻断B7/CD28共刺激通路在小鼠心脏移植中的抗排斥作用

目的探讨RNA干扰(RNAi)技术阻断B7/CD28共刺激通路对小鼠异体心脏移植排斥反应的影响及其机制。方法经体外转录合成针对CD80 mRNA和CD86 mRNA序列特异性小片段干扰RNA(siRNA),转染供者骨髓来源的树突状细胞(DC),半定量逆转录聚合酶链反应、流式细胞仪检测DC转染CD80siRNA、CD86siRNA前后CD80 mRNA和CD86 mRNA的表达水平以及细胞表面CD80及CD86的表达情况。在小鼠异位心脏移植前7d．经静脉给受者输注经siRNA干扰后的DC(干扰DC组),同时设立同种异体对照组、环孢素A(CsA)治疗组(术后皮下注射CsA 5 mg/d)、同系移植对照组和未干扰DC组(移植前输注未转染DC),观察各组移植心脏的存活时间,对移植物的排斥反应进行病理分级,并测定移植物组织中白细胞介素2(IL-2)、γ干扰素(IFN-γ)及IL-10的mRNA表达水平。结果siRNA转染DC后,其CD80 mRNA及CD86 mRNA的表达受到明显抑制,CD80、CD86的阳性率分别由84%和67%下降至35%和30%。与同种异体对照组和未干扰DC组比较,干扰DC组移植心脏存活时间明显延长(P＜0．01),组织排斥反应病理分级显著降低(P <0．01),移植心脏组织中IL-2 mRNA和IFN-γmRNA的表达水平明显降低(P＜0．01),而IL-10 mRNA的表达水平明显升高(P＜0．01)。结论利用RNAi敲减供者骨髓来源的DC表面B7分子的表达,以阻断B7/CD28共刺激通路,具有抑制小鼠心脏移植排斥反应的作用,其机理可能是通过诱导T淋巴细胞无能并使T辅助细胞分化向T_H2型方向偏移。     

RNA干扰阻断BT／CD28共刺激通路在小鼠心脏移植中的抗排斥作用

目的探讨RNA干扰（RNAi）技术阻断B7／CD28共刺激通路对小鼠异体心脏移植排斥反应的影响及其机制。方法经体外转录合成针对CD80 mRNA和CD86 mRNA序列特异性小片段干扰RNA（siRNA），转染供者骨髓来源的树突状细胞（DC），半定量逆转录聚合酶链反应、流式细胞仪检测DC转染CD80siRNA、CD86siRNA前后CD80 mRNA和CD86 mRNA的表达水平以及细胞表面CD80及CD86的表达情况。在小鼠异位心脏移植前7d，经静脉给受者输注经siRNA干扰后的DC（干扰DC组），同时设立同种异体对照组、环孢素A（CsA）治疗组（术后皮下注射CsA 5mg／d）、同系移植对照组和未干扰DC组（移植前输注未转染DC），观察各组移植心脏的存活时间，对移植物的排斥反应进行病理分级，并测定移植物组织中白细胞介素2（IL-2）、γ干扰素（IFN-γ）及IL-10的mRNA表达水平。结果siRNA转染DC后，其CD80 mRNA及CD86 mRNA的表达受到明显抑制，CD80、CD86的阳性率分别由84％和67％下降至35％和30％。与同种异体对照组和未干扰DC组比较，干扰DC组移植心脏存活时间明显延长（P〈0．01），组织排斥反应病理分级显著降低（P〈0．01），移植心脏组织中IL-2 mRNA和IFN-γ mRNA的表达水平明显降低（P〈0．01），而IL-10 mRNA的表达水平明显升高（P〈0．01）。结论利用RNAi敲减供者骨髓来源的DC表面B7分子的表达，以阻断BT／CD28共刺激通路，具有抑制小鼠心脏移植排斥反应的作用，其机理可能是通过诱导T淋巴细胞无能并使T辅助细胞分化向Tn2型方向偏移。     

Treatment of tacrolimus-related adverse effects by conversion to cyclosporine in liver transplant recipients

When tacrolimus side effects persist despite dose reduction, conversion to cyclosporine-based immunosuppression (CyA) is necessary. We characterized tacrolimus side effects that warranted discontinuation of the drug, and outcomes after conversion. Of 388 liver recipients who received tacrolimus as primary immunosuppression, 70 required conversion to CyA. We recorded indication for conversion, whether conversion was early or late after transplantation, tacrolimus dose and trough blood level at conversion, and incidence of rejection after conversion. Conversion was early in 29 patients (41.4 %) and late in 41 (58.6 %). Indications for early conversion were neurotoxicity (20), (insulin-dependent) diabetes mellitus (IDDM) (5), nephrotoxicity (3), gastrointestinal (GI) toxicity (6), and cardiomyopathy (1), and for late conversion were neurotoxicity (15), IDDM (12), nephrotoxicity (3), GI toxicity (5), hepatotoxicity (6), post-transplant lmphoproliferate disease (PTLD) (2), cardiomyopathy (1), hemolytic anemia (1), and pruritis (1). All early-conversion patients showed improvement/resolution of symptoms. Among late-conversion patients, 37 (90.2 %) had improvement/resolution; in 4 (9.8 %), adverse effects persisted. The overall rejection rate was 30 %. Sixty-two patients (88.6 %) are alive with functioning grafts 686 ± 362 days (range, 154–1433 days) after conversion. When tacrolimus side effects are unresponsive to dose reduction, conversion to CyA can be accomplished safely, with no increased risk of rejection and excellent long-term outcome. Received: 26 February 1999/Revised: 5 November 1999/Accepted: 9 November 1999